1. Field of the Invention
This invention is directed to a process for reducing the level of free carbohydrate from a solution of protein-linked carbohydrate (conjugate) and non-linked carbohydrate. In addition, the invention is directed to compositions of the process and to vaccines produced.
2. Description of the Background
Vaccines of protein covalently linked to carbohydrate have proven remarkably successful in inducing an immune response to the carbohydrate moiety. Examples of such vaccines, known as “conjugates” are available for Haemophilus influenzae type b (e.g., ActHib, Hiberix), Neisseria meningiditis types A C W and Y (e.g., Menactra) and S. pneumoniae (e.g., Prevnar, Synflorix) For these vaccines to be effective, it is usually necessary to minimize the amount of non-linked carbohydrate present. The term “carbohydrate” is intended to include polysaccharides, oligosaccharides and other carbohydrate polymers, including monomeric sugars.
Specifications for conjugate vaccines set maximum amounts of free polysaccharide that can be present. In contrast, there is generally no specification for the amount of un conjugated protein. In fact, combination vaccines contain significant amounts of unconjugated protein. For example the five-valent vaccine Pentacel, made by Sanofi-Pasteur, contains Hib PRP polysaccharide conjugated to tetanus toxoid (ActHib) as well as free tetanus toxoid. In any case, removal of the unconjugated protein can usually easily be achieved using size exclusion chromatography, tangential flow filtration or the solid phase method described by U.S. Pat. No. 6,284,250. Thus, it is the reduction in the level of the unconjugated carbohydrate that is critical. This reduction can be difficult to achieve with good efficiency and yield. The absence of a good method for removing the unconjugated polysaccharide reduces the yield, increases the needed effort and increases the cost of manufacturing conjugate vaccines.
Conjugate vaccines tend to be of high molecular weight. First because the carbohydrate component itself may be large, secondly because combining the protein and carbohydrate increases the size, and thirdly because there may be additional cross-linking between the components that further increases the molecular weight.
Chromatography is a common means of purifying biological substances. One means of separating the conjugate from free polysaccharide is size exclusion chromatography (SEC), which separates molecules on the basis of their size, or more precisely, their hydrodynamic radius. SEC is a diffusion-limited, non-adsorptive form of chromatography and suffers from low resolution and capacity. Furthermore, SEC is only successful if there is a significant difference in size molecular weight between the conjugate and the free polysaccharide. Because each is polydisperse, there can be significant overlap in their elution profiles and thus resolution is poor. To obtain material with substantially reduced amounts of free polysaccharide, it is generally necessary to discard part of the conjugate, reducing yields. One solution is to use sized, lower molecular weight polysaccharides and then to crosslink the conjugate sufficiently so that the molecular weight increases enough that it can be separated from the sized PS. This process requires extra processing and additional losses of material.
Chromatography resins (e.g. sorbents, media) consist of porous particles that may be functionalized with charges, ligands and other binding partners. In adsorptive chromatography, substances are bound to the sorbent via these groups. However, to be adsorbed, substances need to enter the pores, a process which is diffusion limited. Most of the surface area of the particles is on the porous interior and large molecules, like conjugates diffuse slowly and due to their size, cannot easily access the pores. These difficulties are further accentuated by the fact that conjugates are poly disperse in size. Thus, some of the smaller conjugate may be able to enter the pores and will chromatograph (i.e., separate) differently than conjugate that does not enter the pores. Due to the fact that the conjugates can generally only access the surface of the particles, their binding capacity for conjugates is severely restricted.
A solution is to use oligosaccharides of a size so that the conjugate formed is still of low enough MW that chromatographic separation can be achieved (e.g., ion exchange). Again, this entails additional processing and losses of material.
The conjugate consists of protein linked to carbohydrate, typically in approximately equal mass. Thus, the conjugate takes on many of the physical properties of the carbohydrate moiety. For example, if the carbohydrate is negatively charged, the conjugate will similarly be negatively charged. This creates a further challenge in removing the free carbohydrate as it makes the chemical properties of the conjugate similar to the free carbohydrate. It is therefore preferable to achieve purification by using properties that are unique to the protein component. Such properties could include an ability to bind to immobilized metal affinity chromatography (IMAC) sorbents. IMAC sorbents can interact with protein histidines, tryptophans and cysteines. However, the metal may leach and need to be subsequently removed. This would be undesirable in the manufacture of vaccines. Another property likely to be unique to the protein is hydrophobicity as carbohydrates are usually much less hydrophobic than proteins. Thus, hydrophobic interaction chromatography (HIC) should be useful for separating conjugate from free polysaccharide. In HIC, a lyotropic salt, such as ammonium sulfate, is added. This salt drives binding of the protein to the hydrophobic surface. Hydrophilic substances, like most carbohydrates, may not bind at all and will be found in the flow through volume. Elution is effected by decreasing the concentration of the lyotropic salt or, less frequently, adding modifiers such as detergents, hydrophobic displacers or organic solvents. More hydrophilic material will elute before more hydrophobic molecules. The principles and practice of HIC is described, for example, in Chapter 7 of Protein Purification 2nd ed., Janson & Ryden (editors), 1998. Conventional HIC chromatography media suffers from poor capacity and poor recovery for use in purifying conjugate vaccines. It is likely that the bulk of the conjugate is unable to enter the pores.
Other methods for separating the conjugate from the free polysaccharide include tangential flow filtration (TFF). In this process, the solution is rapidly passed across a porous membrane with pores of a nominal molecular weight cutoff lower than that of the conjugate and higher than that of the free carbohydrate. The conjugate is retained and the free polysaccharide passes through into the filtrate. This process can be effective if there is a large difference in molecular weight between the conjugate and the free carbohydrate. If they are too close in size, depending on the molecular weight cutoff of the membrane pores, either too much free carbohydrate is retained or too much of the conjugate is found in the filtrate. Furthermore, it has been found that in some cases, even when there is a large difference in size, poor separation and/or recovery is observed. McMasters (U.S. Pat. No. 6,146,902) has claimed that the addition of ammonium sulfate can promote separation by TFF. However, this process was unable to be replicated.
Another nonchromatographic method of separation which takes advantage of the difference in hydrophobicity between the protein and the carbohydrate is the selective precipitation of the protein component with a lyotropic salt such as ammonium sulfate (http://en.wikipedia.org1wiki1Ammonium_sulfate-precipitation). As the protein, whether free or conjugated, is more hydrophobic than the polysaccharide it should precipitate at lower salt concentrations than the free carbohydrate. Thus, the conjugate should precipitate while leaving the carbohydrate in solution. The precipitate is separated by centrifugation and then resuspended. In practice, however, some of the free carbohydrate can become entrapped in the precipitate. Furthermore, there can be significant losses associated with the process.
Thus, there is a significant need for an efficient method to remove the unconjugated carbohydrate from the conjugated carbohydrate in conjugate vaccines.